G-protein coupled receptors (GPCRs) are proteins responsible for transducing a signal within a cell. GPCRs have usually seven transmembrane domains. Upon binding of a ligand to a portion or a fragment of a GPCR, a signal is transduced within the cell that results in a change in a biological or physiological property or behavior of the cell. GPCRs, along with G-proteins, effectors (intracellular enzymes and channels modulated by G-proteins) and beta-arrestins, are the components of a modular signaling system that connects the state of intracellular second messengers to extracellular inputs. GPCR genes and gene products can modulate various physiological processes and are potential causative agents of disease. The GPCRs seem to be of critical importance to both the central nervous system and peripheral physiological processes. The GPCR super family is represented by five families: Family I, receptors typified by rhodopsin and the beta2-adrenergic receptor and currently represented by over 200 unique members; Family II, the parathyroid hormone/calcitonin/secretin receptor family; Family III, the metabotropic glutamate receptor family; Family IV, the cAMP receptor family, important in the chemotaxis and development of D. discoideum; and Family V, the fungal mating pheromone receptor such as STE2. G proteins represent a family of heterotrimeric proteins composed of alpha, beta and gamma subunits, which bind guanine nucleotides. These proteins are usually linked to cell surface receptors (receptors containing seven transmembrane domains) for signal transduction. Indeed, following ligand binding to the GPCR, a conformational-change is transmitted to the G protein, which causes the alpha-subunit to exchange a bound GDP molecule for a GTP molecule and to dissociate from the beta/gamma-subunits. The GTP-bound form of the alpha, beta/gamma-subunits typically functions as an effector-modulating moiety, leading to the production of second messengers, such as cAMP (e.g. by activation of adenyl cyclase), diacylglycerol or inositol phosphates.
Known and uncharacterized GPCRs currently constitute major targets for drug action and development. There are ongoing efforts to identify new GPCRs which can be used to screen for new agonists and antagonists having potential prophylactic and therapeutical properties. More than 300 GPCRs have been cloned to date, excluding the family of olfactory receptors. Mechanistically, approximately 50-60% of all clinically relevant drugs act by modulating the functions of various GPCRs.
Molecular cloning has revealed the existence of at least three human galanin receptor subtypes (Habert-Ortoli et al., Proc. Natl. Acad. Sci. USA 91: 9780-9783, 1994; Burgevin et al., J. Mol. Neurosci. 6: 33-41, 1995; Howard et al., FEBS Letts. 405: 285-290, 1997; Smith et al., J. Biol. Chem. 272: 24612-24616, 1997; Wang et al., Mol. Pharmacol. 52: 337-343, 1997; Wang et al., J. Biol. Chem. 272: 31949-31953, 1997; Ahmad et al., Ann. N.Y. Acad. Sci. 863: 108-119, 1998; Bloomquist et al., Biophys. Res. Commun. 243: 474-479, 1998; Kolakowski et al., J. Neurochem. 71: 2239-2251, 1998; Smith et al., J. Biol. Chem. 273: 23321-23326, 1998). Galanin Receptor 2, alias GALR2 (SEQ NO: 1 (human polynucleotide sequence, FIG. 1); SEQ ID NO: 2 (human amino acid sequence, FIG. 2); SEQ NO: 3 (mouse polynucleotide sequence, FIG. 3); SEQ ID NO: 4 (mouse amino acid sequence, FIG. 4); SEQ NO: 5 (rat polynucleotide sequence, FIG. 5); SEQ ID NO: 6 (rat amino acid sequence, FIG. 6); SEQ NO: 7 (rhesus macaque polynucleotide sequence, FIG. 7); SEQ ID NO: 8 (rhesus macaque amino acid sequence, FIG. 8); SEQ NO: 9 (chimpanzee polynucleotide sequence, FIG. 9), and SEQ ID NO: 10 (chimpanzee amino acid sequence, FIG. 10)), has been described as a GPCR and represents with Galanin Receptor 1 (GALR1) and Galanin Receptor 3 (GALR3), one of the three Galanin receptors.
GALR2 was isolated from rat hypothalamus extract (Howard et al., FEBS Letts. 405: 285-290, 1997; Smith et al., J. Biol. Chem. 272: 24612-24616, 1997; Wang et al., Mol. Pharmacol. 52: 337-343, 1997). This receptor couples to Gi/Go, Gq/G11 or G12 G-protein types, which means that this subtype of galanin receptors can mediate stimulatory as well as inhibitory effects. The distribution of GALR2 is widespread within the CNS but different from that of GALR1. The dorsal root ganglia (DRG) expresses the highest level of GALR2 in the rat (O'Donnell et al., J. Comp. Neurol. 409: 469-481, 1999; Waters and Krause, Neuroscience 95: 265-271, 2000), while low levels of GALR2 mRNA were detected in the rat locus coeruleus (LC) and in the doral raphe nucleus (DRN) region (O'Donnell et al., J. Comp. Neurol. 409: 469-481, 1999). The mouse GALR2 has been reported in the mouse brain but not in the DRN (Hawes et al., J. Comp. Neurol. 479: 410-423, 2004).
The twenty-nine amino-acid, C-amidated neuropeptide galanin was originally isolated from the porcine gut (Tatemoto et al., FEBS Lett. 164: 124-128, 1983) and is widely expressed in both the central and peripheral nervous system. In Human, galanin is based on thirty amino acids with non amidated C-terminal. It has strong inhibitory actions on synaptic transmission by reducing the number of classical neurotransmitters release (Fisone et al, Proc. Natl. Acad. Sci. USA 84: 7339-7343, 1987; Misane et al., Eur. J. Neurosci. 10: 1230-1240, 1998; Pieribone et al., Neurosci. 64: 861-876, 1995; Hokfelt et al., Ann. N.Y. Acad. Sci. 863: 252-263, 1998; Kinney et al., J. Neurosci. 18: 3489-3500, 1998; Zini et al., Eur. J. Pharmacol. 245: 1-7, 1993). These inhibitory actions result in a diverse range of physiological effects, including working memory impairment (Mastropaolo et al., Proc. Natl. Acad. Sci. USA 85: 9841-9845, 1998), long term potentiation (LTP) impairment (Sakurai et al., Neurosci. Lett. 212: 21-24, 1996) and cAMP response element binding (CREB) phosphorylation (Kinney et al., Neurobiol. Learn. Mem. 92: 429-438, 2009), a reduction in hippocampal excitability with a decreased predisposition to seizure activity (Mazarati et al., Brain Res. 589: 164-166, 1992); and a marked inhibition of nociceptive responses in the intact animal and after nerve injury (Wiesenfeld et al., Proc. Natl. Acad. Sci. USA 89, 3: 334-3337, 1992). These neuromodulatory actions of galanin have long been regarded as the principal role played by the peptide in the nervous system. icv. galanin administration to rodents prior to training impaired performance in a wide range of tasks, including spatial learning and passive avoidance (Crawley, Cell. Mol. Life Sci. 65: 1836-1841, 2008), indicating that galanin has a role in short-term working memory and in long-term associative memory processes.
There is a large body of evidence to indicate that injury to many of neuronal systems markedly induces the expression of galanin at both the mRNA and peptide levels. Examples of such lesion studies include the up-regulation of galanin in the DRG following peripheral nerve axotomy (Hokfelt et al., Neurosci. Lett. 83: 217-220, 1987), in the magnocellular secretory neurons of the hypothalamus after hypophysectomy (Villar et al., Neurosci. 36: 181-199, 1990), in the dorsal raphe (DR) and thalamus after removal of the frontoparietal cortex (Cortes et al., Proc. Natl. Acad. Sci. USA 87: 7742-7746, 1990), in the molecular layer of the hippocampus after an entorhinal cortex lesion (Harrison and Henderson, Neurosci. Lett. 266: 41-44, 1999), and in the medial septum (MS) and vertical limb diagonal-band (vdB) after a fimbria formix bundle transection (Brecht et al., Brain Res. Mol. Brain. Res. 48: 7-16, 1997). These studies have led a number of investigators to speculate that galanin might play a cell survival or growth promoting role in addition to its classical neuromodulatory effects. Galanin level is altered in depressive patients (Werner and Coveñas, Int J Neurosci., 120: 455-70, 2010)
The binding of galanin to GALR1 and GALR3 receptors has been shown to inhibit adenyl cyclase (Wang and Gustafson, Drug News Perspect. 11: 458-68, 1998; Habert-Ortoli et al., Proc. Natl. Acad. Sci. USA. 91: 9780-9783, 1994; Smith, J. Biol. Chem. 273: 23321-23326, 1998) by coupling to the inhibitory Gi and/or Go proteins. In contrast, activation of GALR2 with galanin inhibits the release of cAMP by coupling to Gi and/or Go proteins, stimulates phospholipase C and protein kinase C activity by coupling to Gq, hence activating the extracellular signal-regulated kinases (ERK) cascade, and activates Rho by coupling to G12 proteins (Fathi et al., Brain Res Mol Brain Res. 51: 49-59, 1997; Howard et al., FEBS Lett. 405: 285-290, 1997; Wang et al., Mol Pharmacol. 52: 337-343. 1997; Wittau et al., Oncogene 19: 4199-4209, 2000).
The lack of receptor subtype-specific antisera and the paucity of galanin ligands that are receptor subtype-specific, continues to hamper the analysis of the functional roles played by each receptor.
Activation of GALR2 induces cell arrest and apoptosis in head and neck squamous cell carcinoma (HNSCC) (Kanazawa et al., Expert Opin. Ther. Targets. 14: 289-302, 2010).
WO02/096934 discloses a series of galanin agonist compounds which may be used to treat convulsive seizures such as those taking place in epilepsy. There is mention that such compounds could be used for CNS injuries or in open heart surgery to prevent anoxic damage. Wu et al. published information relating to one of these compounds claimed in WO02/096934, named “galnon” (Wu et al., Eur. J. Pharmacol. 482: 133-137, 2003). Galnon equally activates and has agonistic activity to both GALR1 and GALR2. The use of galnon in studies of epilepsy, opioid addiction and feeding behavior has been discussed by different authors (Saar et al. (Proc. Natl. Acad. Sci. U.S.A. 99: 7136-7141, 2002), Zachariou et al. (Proc. Natl. Acad. Sci. U.S.A. 100: 9028-9033, 2003) and Abramov et al. (Neuropeptides 38: 55-61, 2003)).
It is important to note that receptor selectivity of peptidergic galanin receptor ligands is presently a matter of concern. In vitro studies have indicated that M617 (galanin(1-13)-Gln14-bradykinin(2-9)-amide) exhibits 25-fold subtype specificity for GALR1 vs. GALR2, while M871 (galanin-(2-13)-Glu-His-(Pro)3-(Ala-Leu)2-Ala-amide) binds to the GALR2 with a 32-fold higher affinity than to GALR1 (Mazarati et al., J Pharmacol Exp Ther. 318: 700-708, 2006). Both in vitro and in vivo studies support the view that M617 acts as GALR1 agonist, and M871 as a GALR2 antagonist (Mazarati et al., J Pharmacol Exp Ther. 318: 700-708, 2006). Galanin (2-12), AR-M1896, was first described as a GALR2 selective agonist with a nanomolar affinity (Liu et al, Proc Natl Acad Sci USA. 98: 9960-9964, 2001). However, it was subsequently shown that AR-M1896 bind GALR3 receptors with submicromolar affinity in recombinant CHO and COS-7 cell lines, expressing GALR3 receptors (Lu et al, Neuropeptides. 39: 143-146, 2005). Thus, it is important to take into account the binding of this ligand also to the GALR3 receptor when interpreting functional results. Infusion of AR-M1896 into the DR increased serotonin (5-HT) release in the hippocampus (Mazarati et al, J Neurochem 95: 1495-1503, 2005). Thus, it has been speculated that GALR2 receptors in the DR increase serotonergic neurons firing rates and 5-HT release, an effect congruent with the intracellular signaling cascades coupled to GALR2 (Wang et al., Biochemistry 37: 6711-6717, 1998; Branchek et al, Trends Pharmacol Sci 21: 109-117, 2000; Lu et al, Neuropeptides. 39: 143-146, 2005). Interestingly, chronic administration of fluoxetine, a selective serotonine reuptake inhibitor (SSRI) commonly used as an antidepressant, up-regulated GALR2 but not GALR1 in the DR (Lu et al., Proc Natl Acad Sci USA 102: 874-879, 2005). If GALR2 does indeed enhance serotonergic transmission, as suggested by Lu et al., this action of fluoxetine might be one of the mechanisms of its antidepressant effect. Since the GALR2 subtype mediates galanin excitatory actions on neurotransmitter release (Branchek et al, Trends Pharmacol Sci 21: 109-117, 2000), it seems likely that in-vivo AR-M1896 mainly acts as a GALR2 receptor agonist.
Peptide hormones, or neuropeptides, are amino acids string ranging from approximately 3 to 50 residues. More than 100 small peptides have been discovered during the past 30 years (Hokfelt et al., Lancet Neurol. 2: 463-472, 2003). The first neuropeptide, substance P, was identified by Von Euler and Gaddum in 1931, but its exact chemical structure was not described until 1971 (Chang et al., Nat. New. Biol. 232: 86-87, 1971). They are found within a larger protein (a preprohormone), and the production of the mature hormone usually follows specific rules. Preprohormones are secreted proteins, with a signal sequence necessary for the transport of the protein out of the Golgi complex into a secretory vesicle for processing and secretion where the secretion signal is removed, revealing the prohormone. Neuropeptides are synthesized and released from neurons in the central nervous system (CNS) and peripheral nervous system (PNS) and they almost always coexist with classic neurotransmitters (Hokfelt et al., Nature 284: 525-521, 1980). Neuropeptides can function as neurotransmitters, hormones, and growth factors and their actions are mediated through GPCRs.
In general, hormones are surrounded by basic residues pair, i.e. Arg-Arg, Arg-Lys, Lys-Arg, or Lys-Lys (cleavage sites), which are found directly adjacent to the putative hormone. These double basic residues act as recognition sites for the processing enzymes, usually serine proteases that cleave the prohormone to liberate the active (mature) peptide. In many cases, there is more than a single active peptide within one precursor protein. Another common feature shared by neuropeptides is the presence of an amide group at the C-terminal (instead of the usual carboxylic acid), which provides protection against enzymatic degradation and is required for biological activity. Much evidence indicates that neuropeptides are of particular importance when the nervous system is challenged, e.g. by stress, traumatic events, injury, or drug abuse, modulating the activity of co-expressed neurotransmitters (Hokfelt et al., Lancet Neurol. 2: 463-472, 2003). Binding affinities of neuropeptides are commonly in the nanomolar range, which is thousand-fold or higher than the classical neurotransmitters. Consequently the selectivity of neuropeptide receptors is high, possibly making pharmacological interventions with modulators less prone to side-effects. These features and the large number of neuropeptides and neuropeptide receptors provide many opportunities for the discovery of new drug targets to treat CNS disorders.
A number of neuropeptides have been identified as potential targets for the antidepressant drugs development. For instance, it has been reported that neurokinin 1 antagonist is as efficient as a SSRI in major depression disorder (Kramer et al., Science 281: 1640-1645, 1998). Other neuropeptides implicated in mood regulation and anxiety are for example neuropeptide Y (NPY), corticotrophin releasing factor (CRF), nociceptin and galanin.
Neuropeptide Q, alias Spexin, is a hormone recently discovered by Mirabeau et al. (Genome Res., 17: 320-327, 2007). The authors developed a Hidden Markov Model (HMM) based on algorithm searches that integrates several peptide hormone sequence features to identify novel peptide hormones. To examine whether the predicted Neuropeptide Q could moderate smooth muscle contractility, a synthetic amidated Neuropeptide Q, NWTPQAMLYLKGAQ-amide, was tested by Mirabeau et al. in stomach explants contractility assay. The Neuropeptide Q dose-dependently induced contraction of stomach muscle with an EC50 of 0.75 μM.
A peptide with a similar sequence has been discovered by Hsueh et al. and named cosmedin B (US20050221359). Cosmedin B interperitoneal treatment suppressed gastric emptying activity in a dose-dependant manner. In an organ contraction assay using rat ileal tissue strips, incubation with cosmedin B produced a concentration dependent muscle contraction. The normalization procedure demonstrated that the magnitude of contraction induced by cosmedin B was comparable to that mediated by the muscarinic receptor stimulated by 5-methyl furmethide.